Lecithin:cholesterol acyltransferase (LCAT), which synthesizes the bulk of cholesteryl esters, and cholesteryl ester transfer protein (CETP), which transfers these from high (HDL) to low (LDL) and very low (VLDL) density lipoproteins, are two major catalytic factors of human plasma cholesterol metabolism and may play a major role in determining the total concentration and lipoprotein distribution of cholesterol in plasma. CETP activity can be inhibited by a circulating inhibitory protein, recently identified as apolipoprotein D (apo D). The activity of these factors under different physiological and pathological conditions is in large part determined posttranslationally. Site-directed mutagenesis will be used to localize functionally important residues in each protein. In LCAT, the catalytic mechanism will be studied by identifying the location of the serine, histidine, and aspartate residues likely to make up a catalytic triad in this enzyme. We will test the hypothesis that the lipid specificity of LCAT is determined by the mean hydrophobic index of the residues surrounding serine-181 of the primary sequence. We will also test the hypothesis that a neighboring sequence plays a key role in the binding of LCAT to its high density lipoprotein substrate. Finally we will determine the location of the N-linked carbohydrate residues, which have a major effect of LCAT catalytic rate. In CETP, we will test the hypothesis that cholesteryl ester and triglyceride bind to distinct but neighboring sites at the C-terminal end of the primary sequence, and will make both deletions and point mutations to localize and define the residues involved. We will use first covalent chemical modification and then mutagenesis to define this site. Finally, we will investigate the location and role of the N-linked carbohydrate residues, which are required for catalytic activity. For the CETP inhibitor protein, we will confirm its identity with apo D, and then use site-directed mutagenesis (assisted by sequence similarities to related lipid-binding proteins) to define the regions responsible for ligand binding and for the ability to displace CETP from HDL. These studies apply molecular biology techniques to study the regulation of proteins whose activity can have major effects on tissue and plasma cholesterol content.